1. Field of the Invention
This invention relates to micromachined reference electrodes, for example, for use in miniaturized electrochemical sensors, and to methods for fabricating such reference electrodes and electrochemical sensors, for example, as a part of a microfluidic system.
2. Description of the Related Technology
Electrochemical measurements allow for inexpensive detection of a wide variety of (bio-)chemical compounds in solution. The reference electrode is one of the main parts of an electrochemical cell. The reference electrode, from which no current is drawn, has a stable, constant potential. There are two kinds of electrochemical sensors that need a reference electrode: amperometric sensors and potentiometric sensors. Amperometric sensors comprise a sensing electrode or working electrode, a counter electrode and a reference electrode. Potentiometric sensors comprise an ion-selective electrode (ISE) as a working electrode and a reference electrode.
In the case of amperometric sensors, a current is generated between the sensing electrode and the counter electrode when a compound that needs to be detected is oxidized or reduced on the sensing electrode. The current depends on the concentration of the compound. The current also depends on the sensing electrode potential, which is generated by an external power source. The sensing electrode is defined relative to the reference electrode potential. Therefore, accurate and reproducible current measurements can only be obtained if a stable reference electrode potential is available.
In the case of potentiometric sensors, the measured parameter is the open circuit voltage (OCV) between the ISE and the reference electrode. The ISE shows a high selectivity towards certain compounds, where the OCV changes as a function of the concentration of these compounds.
As can be deduced from the previous two paragraphs, the reference electrode is a crucial part of an electrochemical sensor. An unstable reference electrode in an amperometric sensor results in a shift of the sensing electrode potential, which affects the output current. The situation is even more detrimental in potentiometric sensors, where any fluctuation of the reference electrode potential is fully reflected in the output signal. An instable reference electrode inevitably results in malfunctioning of the electrochemical sensor.
Existing systems often use an external, macroscopic reference electrode or a quasi-reference electrode, which is directly exposed to the sample solution. External, macroscopic reference electrodes typically have a size of approximately 10 cm. An Ag/AgCl electrode, among other materials, is often used in an external, macroscopic reference electrode, where the Ag/AgCl electrode is often immersed in a solution that contains 3 molar KCl and saturated AgCl. The voltage of an Ag/AgCl electrode depends on the chloride concentration of the solution in contact with the electrode. Therefore, if a constant voltage is needed, the chloride concentration needs to be kept constant. In order to do that, the KCl solution in the reference electrode is physically separated from the sample solution by means of an electrolyte bridge or a porous, ceramic plug. As a result, the chloride concentration does not change significantly. At the same time, there is contact with the external solution that contains the other electrodes trough the electrolyte bridge or the porous, ceramic plug. However, such a macroscopic reference electrode configuration that is based on glass tubes is difficult to miniaturize. Moreover, a liquid-junction potential is established across a porous, ceramic plug, which adds up to the reference potential leading to a misestimating of the sensing electrode potential.
Various miniature electrochemical sensors have been developed. The fabrication technologies for these devices and their performance have advanced sufficiently to realize their commercialization. However, one of the main problems that have restricted their application is the unavailability of a reliable and durable miniature reference electrode. Miniaturized electrochemical sensors often contain a quasi-reference electrode. In this case, the electrode, e.g. Ag/AgCl electrode, is not submerged in a separate KCl solution but in the sample solution together with the other electrodes. Two stability problems are inherent to this approach. First, the KCl concentration, which is related to the reference electrode voltage, is not constant. Second, interfering redox reactions with the analyte and other substances may take place at the reference electrode.
Microfabrication of reference electrodes can be done by micromachining of KCl solution reservoirs in silicon, for example, by means of KOH etching. In some cases, a plug may be needed to increase the lifetime, although this may lead to an increase of the junction potential. For example, pyramidal reservoirs can be created by KOH etching leading to a pinhole at one side, where the pinhole is in contact with the sample solution during operation. In general, it is difficult to use this approach for the integration of a larger microfluidic system due to constraints of the KOH etching process. For example, there are constraints to the shapes of reservoirs and channels that can be fabricated by means of KOH etching. Moreover, it is only possible to etch silicon in a KOH solution. Silicon is a relatively expensive material and it is preferably replaced by other materials such as glass and polymers.
The lifetime of a liquid-junction reference electrode is related to the decrease of the electrolyte concentration in the reference electrode reservoir, for example, as a result of effusion and outflow from the reference electrode reservoir, leading to a change of the reference potential. In order to slow down the effusion of KCl from the reference electrode, the pinhole can be closed by a porous silicon membrane acting as a diffusion barrier, for example, as reported by R. L. Smith and D. C. Scott in “An integrated sensor for electrochemical measurements”, IEEE Trans. Biomed. Eng. 33, 83 (1986). However, process integration of porous silicon can be difficult and thus the reproducibility of this approach is lacking. Diffusion barriers other than porous silicon can be used such as porous silica glass and polymers. The fabrication and integration of porous materials is quite complex because it requires wet chemical processes that are not easily scaled up. Alternatively, instead of using a diffusion barrier, the pyramidal reservoir can be filled with a hydrogel solution to avoid leaching out of the KCl solution, for example, as reported by A. van den Berg et al. in “A micro-volume open liquid-junction reference electrode for pH-ISFETs”, Sensors and Actuators B 1, 425 (1990). However, using this approach the reference electrode potential drift could not be reduced below 0.1 mV·h−1. In general, the use of a porous plug or a hydrogel to reduce the diffusion of ions may increase the liquid-junction potential in an unpredictable manner. Moreover, the response time to a change in the sample solution can increase as a result of the slower ion diffusion in the porous plug or the hydrogel.
More advanced techniques for the microfabrication of liquid-junction reference electrodes, leading to the creation of larger microfluidic systems, have been reported. For example, a method is described in U.S. Pat. No. 6,419,809 for fabricating a complete reference electrode in silicon using microfabrication techniques. The microfluidic part comprises a reservoir, a channel and a pinhole. However, a reliable diffusion barrier was only obtained by closing the pinhole with a polymer plug. Typical lifetime values for the systems described in U.S. Pat. No. 6,419,809 are in the order of one day. Moreover, a polymer plug may influence the junction potential in an unpredictable manner and complicates the fabrication process. The lifetime of the reference electrode is also dependent on the durability of the internal electrode, usually an Ag/AgCl electrode, and not only on the stability of the KCl concentration. Other internal electrode materials and electrolytes can also be used if this improves the reference electrode lifetime.